Herbal medicinal composition and extract thereof for inhibiting growth of cancer cells

ABSTRACT

The present invention relates to herbal medicinal compositions and extracts thereof for inhibiting growth of cancer cells. One of the examples described in the present invention comprises Forsythiae fructus, Menthae Herba, Gardeniae Fructus, Scutellariae Radix, Lophatheri Folium, Glycyrrhizae Radix, Rhei Rhizoma, Na 2 SO 4 , and  Atractylodis Rhizoma  . The residual examples are described herein.

This application is a divisional application of pending U.S. patentapplication Ser. No. 12/923,846, filed Oct. 12, 2010 (of which theentire disclosure of the pending, prior application is herebyincorporated by reference).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to herbal medicinal compositions andextracts thereof for inhibiting growth of cancer cells and, moreparticularly, to herbal medicinal compositions and extracts thereof forinhibiting growth of lung cancer cells.

2. Description of Related Art

Cancers have been one of ten leading causes of death in the lastcontinuous 27 years. The main cause of cancer is abnormality of cellswhich undergo self-division continuously to form more and more abnormalcells, i.e., cancers.

In common tumor cells, some cancer cells have characteristics of stemcells. Although such cancer cells are in a small number, they canundergo cell division and differentiation continuously, similar to stemcells, and thus are so called “cancer stem cells”. Since cancer stemcells have extremely high drug resistance, it is difficult forchemotherapeutic agents of modern (Western) medicine to exterminatethem. Accordingly, it is often heard that cancer recurrence happens inmany patients post-chemotherapy. In addition, standard therapiescurrently known in biomedical science are still unable to kill suchcancer stem cells.

Furthermore, surgical operations, radiotherapies, chemotherapies,hormone therapies, biological therapies, and so on in modern medicalscience may incur strongly unfavorable side effects to patients.Therefore, it is a significant breakthrough if a cancer can be treatedby a therapy which is relatively gentle and able to inhibit developmentof cancer stem cells.

Currently, people believe that the use of Chinese herbal medicine totreat patients is both a gentle therapy and highly acceptable incommerce. Hence, if a developed herbal medicinal composition isevidenced to inhibit cancer cells or block division of cancer stemcells, it will be considerably helpful to the treatment of cancers.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a herbal medicinalcomposition and an extract thereof for inhibiting growth of cancer cellssuch as lung cancer cells.

To achieve the object, one aspect of the present invention provides aherbal medicinal composition for inhibiting cancer cells including:Gentianae Scabrae Radix (Gentiana scabra Bunge), Scutellariae Radix(Scutellaria baicalensis Georgi), Gardeniae Fructus (Gardeniajasminoides Ellis), Anelicae Sinensis Radix (Angelica sinensis Diels),Rehmanniae Radix (Rehmannia glutinosa Libosch. f. hueichingensis (Chaoet Schih) Hsiao), Hocquartiae Caulis (Hocquartia manshuriensis (Kom.)Nakai), Bupleuri Radix (Bupleurum falcatum Linne), Plantaginis Semen(Plantago asiatica Linne), Atractylodis Rhizoma (Atractylodes lancea DeCandolle), Alismatis Rhizoma (Alisma plantago-aquatica L. var. orientaleSAM.), and Glycyrrhizae Radix (Glycyrrhiza uralensis Fischer et DC).This herbal medicinal composition is extracted, i.e., Gentianae ScabraeRadix, Scutellariae Radix, Gardeniae Fructus, Anelicae Sinensis Radix,Rehmanniae Radix, Hocquartiae Caulis, Bupleuri Radix, Plantaginis Semen,Atractylodis Rhizoma, Alismatis Rhizoma, and Glycyrrhizae Radix aremixed, extracted with water under heating, and then filtrated to removethe dreg thereof. A herbal medicinal extract can be obtained and it alsocan inhibit growth of cancer cells.

Preferably, the Gentianae Scabrae Radix is comprised in an amount of1.5-6 parts by weight, the Scutellariae Radix is comprised in an amountof 1.5-6 parts by weight, the Gardeniae Fructus is comprised in anamount of 1.5-6 parts by weight, the Anelicae Sinensis Radix iscomprised in an amount of 1.5-6 parts by weight, the Rehmanniae Radix iscomprised in an amount of 1.5-6 parts by weight, the Hocquartiae Caulisis comprised in an amount of 1.5-6 parts by weight, the Bupleuri Radixis comprised in an amount of 1.5-6 parts by weight, the PlantaginisSemen is comprised in an amount of 1.5-6 parts by weight, theAtractylodis Rhizoma is comprised in an amount of 2-8 parts by weight,the Alismatis Rhizoma is comprised in an amount of 2-8 parts by weight,and the Glycyrrhizae Radix is comprised in an amount of 2.5-10 parts byweight.

More preferably, the Gentianae Scabrae Radix is comprised in an amountof 2-4 parts by weight, the Scutellariae Radix is comprised in an amountof 2-4 parts by weight, the Gardeniae Fructus is comprised in an amountof 2-4 parts by weight, the Anelicae Sinensis Radix is comprised in anamount of 2-4 parts by weight, the Rehmanniae Radix is comprised in anamount of 2-4 parts by weight, the Rehmanniae Radix is comprised in anamount of 2-4 parts by weight, the Bupleuri Radix is comprised in anamount of 2-4 parts by weight, the Plantaginis Semen is comprised in anamount of 2-4 parts by weight, the Atractylodis Rhizoma is comprised inan amount of 3-6 parts by weight, the Alismatis Rhizoma is comprised inan amount of 3-6 parts by weight, and the Glycyrrhizae Radix iscomprised in an amount of 4-7 parts by weight.

Another aspect of the present invention provides a herbal medicinalcomposition for inhibiting cancer cells including: Forsythiae fructus(Forsythia suspense (Thunb.) Vahl), Menthae Herba (Mentha haploclyxBRIQ.), Gardeniae Fructus, Scutellariae Radix, Lophatheri Folium(Lophatherum gracile Brongniart), Glycyrrhizae Radix, Rhei Rhizoma(Rheum palmatum Linne), Na₂SO₄, and Atractylodis Rhizoma. This herbalmedicinal composition is extracted, i.e., Forsythiae fructus, MenthaeHerba, Gardeniae Fructus, Scutellariae Radix, Lophatheri Folium,Glycyrrhizae Radix, Rhei Rhizoma, Na₂SO₄, and Atractylodis Rhizoma aremixed, extracted with water under heating, and then filtrated to removethe dreg thereof. A herbal medicinal extract can be obtained and it caninhibit growth of cancer cells.

Preferably, the Forsythiae fructus is comprised in an amount of 1.5-6parts by weight, the Menthae Herba is comprised in an amount of 1.5-6parts by weight, the Gardeniae Fructus is comprised in an amount of1.5-6 parts by weight, the Scutellariae Radix is comprised in an amountof 1.5-6 parts by weight, the Lophatheri Folium is comprised in anamount of 1.5-6 parts by weight, the Glycyrrhizae Radix is comprised inan amount of 1.5-6 parts by weight, the Rhei Rhizoma is comprised in anamount of 0.5-2 parts by weight, the Na₂SO₄ is comprised in an amount of0.5-2 parts by weight, and the Atractylodis Rhizoma is comprised in anamount of 2-8 parts by weight.

More preferably, the Forsythiae fructus is comprised in an amount of 2-4parts by weight, the Menthae Herba is comprised in an amount of 2-4parts by weight, the Gardeniae Fructus is comprised in an amount of 2-4parts by weight, the Scutellariae Radix is comprised in an amount of 2-4parts by weight, the Lophatheri Folium is comprised in an amount of 2-4parts by weight, the Glycyrrhizae Radix is comprised in an amount of 2-4parts by weight, the Rhei Rhizoma is comprised in an amount of 0.75-1.5parts by weight, the Na₂SO₄ is comprised in an amount of 0.75-1.5 partsby weight, and the Atractylodis Rhizoma is comprised in an amount of 3-6parts by weight.

Still another aspect of the present invention provides a herbalmedicinal composition for inhibiting cancer cells including: AnelicaeSinensis Radix, Ligustici Rhizoma (Ligusticum chuanxiong Hortorum),Paeoniae (Ovatae) Radix Rubra (Paeonia veitchii Lynch.), RehmanniaeRadix, Glycyrrhizae Radix, Scutellariae Radix, Chinese WolfberryRoot-bark (Lycium chinense Mill. Root), Peony Root-bark (Paeoniasuffruticosa Andr. Root), and Atractylodis Rhizoma. This herbalmedicinal composition is extracted, i.e., Anelicae Sinensis Radix,Ligustici Rhizoma, Paeoniae (Ovatae) Radix Rubra, Rehmanniae Radix,Glycyrrhizae Radix, Scutellariae Radix, Chinese Wolfberry Root-bark,Peony Root-bark, and Atractylodis Rhizoma are mixed, extracted withwater under heating, and then filtrated to remove the dreg thereof. Aherbal medicinal extract can be obtained and it can inhibit growth ofcancer cells.

Preferably, the Anelicae Sinensis Radix is comprised in an amount of1.5-6 parts by weight, the Ligustici Rhizoma is comprised in an amountof 1.5-6 parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprisedin an amount of 1.5-6 parts by weight, the Rehmanniae Radix is comprisedin an amount of 1.5-6 parts by weight, the Glycyrrhizae Radix iscomprised in an amount of 1.5-6 parts by weight, the Scutellariae Radixis comprised in an amount of 1.5-6 parts by weight, the ChineseWolfberry Root-bark is comprised in an amount of 2.5-10 parts by weight,the Peony Root-bark is comprised in an amount of 2.5-10 parts by weight,and the Atractylodis Rhizoma is comprised in an amount of 2-8 parts byweight.

More preferably, the Anelicae Sinensis Radix is comprised in an amountof 2-4 parts by weight, the Ligustici Rhizoma is comprised in an amountof 2-4 parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprisedin an amount of 2-4 parts by weight, the Rehmanniae Radix is comprisedin an amount of 2-4 parts by weight, the Glycyrrhizae Radix is comprisedin an amount of 2-4 parts by weight, the Scutellariae Radix is comprisedin an amount of 2-4 parts by weight, the Chinese Woliberry Root-bark iscomprised in an amount of 4-7 parts by weight, the Peony Root-bark iscomprised in an amount of 4-7 parts by weight, and the AtractylodisRhizoma is comprised in an amount of 2-8 parts by weight.

Yet a further aspect of the present invention provides a herbalmedicinal composition for inhibiting cancer cells including: AnelicaeSinensis Radix, Ligustici Rhizoma, Peony Root (Paeonia lacqlora Pall.),Rehmanniae Radix, Atractylodis Rhizoma (Atractylodes ovata DeCanndolle), Glycyrrhizae Radix, Dipsaci Radix (Dipsacus asper Wall.),Eucommia Bark (Eucommia ulmoides Oliv), Scutellariae Radix, DioscoreaeRhizoma (Dioscorea opposites Thunb.), and Asiatic Wormwood (Artemisiaargyi Levi. et Vant.). This herbal medicinal composition is extracted,i.e., Anelicae Sinensis Radix, Ligustici Rhizoma, Peony Root, RehmanniaeRadix, Atractylodis Rhizoma, Glycyrrhizae Radix, Dipsaci Radix, EucommiaBark, Scutellariae Radix, Dioscoreae Rhizoma, and Asiatic Wormwood. aremixed, extracted with water under heating, and then filtrated to removethe dreg thereof A herbal medicinal extract can be obtained and it caninhibit growth of cancer cells.

Preferably, the Anelicae Sinensis Radix is comprised in an amount of1.5-6 parts by weight, the Ligustici Rhizoma is comprised in an amountof 1.5-6 parts by weight, the Peony Root is comprised in an amount of1.5-6 parts by weight, the Rehmanniae Radix is comprised in an amount of1.5-6 parts by weight, the Atractylodis Rhizoma is comprised in anamount of 1.5-6 parts by weight, the Glycyrrhizae Radix is comprised inan amount of 1.5-6 parts by weight, the Dipsaci Radix is comprised in anamount of 1.5-6 parts by weight, the Eucommia Bark is comprised in anamount of 1.5-6 parts by weight, the Scutellariae Radix is comprised inan amount of 1.5-6 parts by weight, the Dioscoreae Rhizoma is comprisedin an amount of 2.5-10 parts by weight, and the Asiatic Wormwood. iscomprised in an amount of 2.5-10 parts by weight.

More preferably, the Anelicae Sinensis Radix is comprised in an amountof 2-4 parts by weight, the Ligustici Rhizoma is comprised in an amountof 2-4 parts by weight, the Peony Root is comprised in an amount of 2-4parts by weight, the Rehmanniae Radix is comprised in an amount of 2-4parts by weight, the Atractylodis Rhizoma is comprised in an amount of2-4 parts by weight, the Glycyrrhizae Radix is comprised in an amount of2-4 parts by weight, the Dipsaci Radix is comprised in an amount of 2-4parts by weight, the Eucommia Bark is comprised in an amount of 2-4parts by weight, the Scutellariae Radix is comprised in an amount of 2-4parts by weight, the Dioscoreae Rhizoma is comprised in an amount of 4-7parts by weight, and the Asiatic Wormwood. is comprised in an amount of4-7 parts by weight.

Still another aspect of the present invention provides a herbalmedicinal composition for inhibiting cancer cells including: CinnamomumRamulus (Cinnamomum cassia Presl.), Paeoniae (Ovatae) Radix Rubra,Glycyrrhizae Radix, Zingiberis Rhizoma (Zingiber officinale Roscoe),Scutellariae Radix, and Jujubae Fructus (Ziziphus jujuba Mill.). Thisherbal medicinal composition is extracted, i.e., Cinnamomum Ramulus,Paeoniae (Ovatae) Radix Rubra, Glycyrrhizae Radix, Zingiberis Rhizoma,Scutellariae Radix, and Jujubae Fructus. are mixed, extracted with waterunder heating, and then filtrated to remove the dreg thereof. A herbalmedicinal extract can be obtained and it can inhibit growth of cancercells.

Preferably, the Cinnamomum Ramulus is comprised in an amount of 2.5-10parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprised in anamount of 2.5-10 parts by weight, the Glycyrrhizae Radix is comprised inan amount of 2.5-10 parts by weight, the Zingiberis Rhizoma is comprisedin an amount of 2.5-10 parts by weight, the Scutellariae Radix iscomprised in an amount of 1.5-6 parts by weight, and the JujubaeFructus. is comprised in an amount of 2.5-10 parts by weight.

More preferably, the Cinnamomum Ramulus is comprised in an amount of 4-7parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprised in anamount of 4-7 parts by weight, the Glycyrrhizae Radix is comprised in anamount of 4-7 parts by weight, the Zingiberis Rhizoma is comprised in anamount of 4-7 parts by weight, the Scutellariae Radix is comprised in anamount of 2-4 parts by weight, and the Jujubae Fructus. is comprised inan amount of 4-7 parts by weight.

Still a further aspect of the present invention provides a herbalmedicinal composition for inhibiting cancer cells including: CimicifugaeRhizoma (Cimicifuga heracleifolia Kam), Rehmanniae Radix, Paeoniae(Ovatae) Radix Rubra, Peony Root-bark, and Scutellariae Radix. Thisherbal medicinal composition is extracted, i.e., Cimicifugae Rhizoma,Rehmanniae Radix, Paeoniae (Ovatae) Radix Rubra, Peony Root-bark, andScutellariae Radix are mixed, extracted with water under heating, andthen filtrated to remove the dreg thereof. A herbal medicinal extractcan be obtained and it can inhibit growth of cancer cells.

Preferably, the Cimicifugae Rhizoma is comprised in an amount of 1-4parts by weight, the Rehmanniae Radix is comprised in an amount of7.5-30 parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprisedin an amount of 5-20 parts by weight, the Peony Root-bark is comprisedin an amount of 5-20 parts by weight, and the Scutellariae Radix iscomprised in an amount of 5-20 parts by weight.

More preferably, the Cimicifugae Rhizoma is comprised in an amount of1.5-3 parts by weight, the Rehmanniae Radix is comprised in an amount of10-20 parts by weight, the Paeoniae (Ovatae) Radix Rubra is comprised inan amount of 8-15 parts by weight, the Peony Root-bark is comprised inan amount of 8-15 parts by weight, and the Scutellariae Radix iscomprised in an amount of 8-15 parts by weight.

Besides, in the above mentioned herbal medicinal composition and theherbal medicinal extract thereof, the temperature and time of theheating, the amount of used water, and condensing ratio of the extractare not specifically limited. Preferably, the heating is to heat thewater to 90-100° C. for 60-90 mins, the water is used in an amount of5-15 times the total weight of the mixture, and the extract is condensedto ½-¼ weight of the water by heating.

Other objects, advantages, and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the relation between the survival rate ofA549 lung cancer cells and the time of treating A549 lung cancer cellswith the extract of Example 5 of the present invention, in which ***represents p<0.001 after Student's t test;

FIG. 2 is a diagram showing the rates of A549 lung cancer cells inG0/G1, S, and G2/M phases posterior to being treated with the extract ofExample 5 of the present invention, in which *, **, and *** representsp<0.05, 0.01, and 0.001 after Student's t test, respectively; and

FIG. 3 is a diagram showing the rate of A549 lung cancer cells in G0phase posterior to being treated with the extract of Example 5 of thepresent invention, in which *** represents p<0.001 after Student's ttest.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the specific embodiments illustrating the practice of thepresent invention, a person having ordinary skill in the art can easilyunderstand other advantages and efficiency of the present inventionthrough the content disclosed therein. The present invention can also bepracticed or applied by other variant embodiments. Many other possiblemodifications and variations of any detail in the present specificationbased on different outlooks and applications can be made withoutdeparting from the spirit of the invention.

EXAMPLE 1

Gentianae Scabrae Radix (11.25 g), Scutellariae Radix (11.25 g),Gardeniae Fructus (11.25 g), Anelicae Sinensis Radix (11.25 g),Rehmanniae Radix (11.25 g), Rehmanniae Radix (11.25 g), Bupleuri Radix(11.25 g), Plantaginis Semen (11.25 g), Atractylodis Rhizoma (15 g),Alismatis Rhizoma (15 g), and Glycyrrhizae Radix (18.75 g) all were cutinto slices, respectively. A mixture containing the abovementionedherbal medicinal materials was extracted with water (1200 g) underheating to 90° C. or more for 60-90 minutes, condensed into 400 g, andthen filtrated to remove the dreg thereof to afford an extract.

EXAMPLE 2

Forsythiae fructus (11.25 g), Menthae Herba (11.25 g), Gardeniae Fructus(11.25 g), Scutellariae Radix (11.25 g), Lophatheri Folium (11.25 g),Glycyrrhizae Radix (11.25 g), Rhei Rhizoma (3.75 g), Na₂SO₄ (3.75 g),and Atractylodis Rhizoma (15 g) all were cut into slices, respectively.A mixture containing the abovementioned herbal medicinal materials wasextracted with water (1200 g) under heating to 90° C. or more for 60-90minutes, condensed into 400 g, and then filtrated to remove the dregthereof to afford an extract.

EXAMPLE 3

Anelicae Sinensis Radix (11.25 g), Ligustici Rhizoma (11.25 g), Paeoniae(Ovatae) Radix Rubra (11.25 g), Rehmanniae Radix (11.25 g), GlycyrrhizaeRadix (11.25 g), Scutellariae Radix (11.25 g), Chinese WolfberryRoot-bark (18.75 g), Peony Root-bark (18.75 g), and Atractylodis Rhizoma(15 g) all were cut into slices, respectively. A mixture containing theabovementioned herbal medicinal materials was extracted with water (1200g) under heating to 90° C. or more for 60-90 minutes, condensed into 400g, and then filtrated to remove the dreg thereof to afford an extract.

EXAMPLE 4

Anelicae Sinensis Radix (11.25 g), Ligustici Rhizoma (11.25 g), PeonyRoot (11.25 g), Rehmanniae Radix (11.25 g), Atractylodis Rhizoma (11.25g), Glycyrrhizae Radix (11.25 g), Dipsaci Radix (11.25 g), Eucommia Bark(11.25 g), Scutellariae Radix (11.25 g), Dioscoreae Rhizoma (18.75 g),and Asiatic Wormwood. (18.75 g) all were cut into slices, respectively.A mixture containing the abovementioned herbal medicinal materials wasextracted with water (1200 g) under heating to 90° C. or more for 60-90minutes, condensed into 400 g, and then filtrated to remove the dregthereof to afford an extract.

EXAMPLE 5

Cinnamomum Ramulus (18.75 g), Paeoniae (Ovatae) Radix Rubra (18.75 g),Glycyrrhizae Radix (18.75 g), Zingiberis Rhizoma (18.75 g), ScutellariaeRadix (11.25 g), and Jujubae Fructus. (18.75 g) all were cut intoslices, respectively. A mixture containing the abovementioned herbalmedicinal materials was extracted with water (1200 g) under heating to90° C. or more for 60-90 minutes, condensed into 400 g, and thenfiltrated to remove the dreg thereof to afford an extract.

EXAMPLE 6

Cimicifugae Rhizoma (7.5 g), Rehmanniae Radix (56.25 g), Paeoniae(Ovatae) Radix Rubra (37.5 g), Peony Root-bark (37.5 g), andScutellariae Radix (37.5 g) all were cut into slices, respectively. Amixture containing the abovementioned herbal medicinal materials wasextracted with water (1200 g) under heating to 90° C. or more for 60-90minutes, condensed into 400 g, and then filtrated to remove the dregthereof to afford an extract.

TEST EXAMPLE

Researches have directed that cancer stem cells are those exhibitingcharacteristics of stem cells in cancer cells. Therefore, if treatmentfocuses on common cancer cells, cancer stem cells may still survive in atreated tumor. Owing to characteristics of stem cells, the cancer stemcells which survive still can differentiate into commons cancer cellsand even form a malignant tumor, resulting in cancer recurrence aftertreatment. However, if treatment can focus on cancer stem cells, tumorshave no survivor of cancer stem cells which can differentiate to supplythe number of common cancer cells, and thus can be inhibited.

According to the reported researches, some researchers have isolatedcancer stem cells having characteristics of stem cells from A549 lungcancer cells. Therefore, in the following Test Examples of the presentinvention, the extracts from the herbal medicinal composition of thepresent invention were analyzed for the survival rate of A549 lungcancer cells. In addition, the cell cycle of cancer cells was checked byflow cytometry to determine whether it was blocked by the extracts ofthe present invention. The influence of the extracts of the presentinvention on promotion of cell apoptosis was also confirmed by stainingmethods. Besides, flow cytometry and two-color fluorescence stainingwere performed to determine the cytotoxicity of the extracts of thepresent invention in cancer cells.

Test Example 1 Test for Survival Rate of Cells

A549 lung cancer cells were treated with the extracts of Examples 1-6respectively with a concentration of 10%, 20%, and 100% for 72 hours,and then their survival rates were determined by an MTT assay. Theresults are shown in the following Table 1.

TABLE 1 Survival Rate Extract 5 μl 10 μl 50 μl Pure Water 45 μl 40 μl 0μl Example 1 96% 96% 35% Example 2 89% 83% 29% Example 3 24% 11%  5%Example 4 107%  80%  7% Example 5 97% 77% 41% Example 6 76% 70% 12%Survival rate (%) = average absorbance of experimental groups/averageabsorbance of control group Control group: treated with pure water butwithout the extracts

As listed in Table 1, the survival rates of A549 cancer cells treatedafter 72 hours reduce as the concentration of the extracts increase.

Test Example 2 Influence of Treating Time on the Survival Rate of theCancer Cells

According to the result of Test Example 1, it can be seen that a halfmaximal inhibitory dose of the extract of Example 5 is 35 μl. Therefore,A549 lung cancer cells were treated with the half maximal inhibitorydose (35 μl) for 24, 48, and 72 hours, and were analyzed by an MTT assayto check the survivors thereof. The results are shown in the FIG. 1.

As shown in FIG. 1, compared with the control group of A549 lung cancercells untreated with the extract of Example 5, the experimental groupsthereof treated with the extract of Example 5 for 24, 48, and 72 hoursall have reduced survival rates. Furthermore, as the treating timeextends, the survival rates have more significant diversity.

Test Example 3 Test of Checking Cell Cycle Block

(1) PI Staining

A549 lung cancer cells were treated with the half maximal inhibitorydose (35 μl) of the extract of Example 5 for 24, 48, and 72 hours, andthen underwent PI staining followed with flow cytometry to determine DNAlevel of the lung cancer cells. After analysis of statistics, the resultis shown in FIG. 2. In FIG. 2, G0/G1, S, G2/M on the horizontal axisrepresent specific phases in a cell cycle, and the vertical axisrepresents cell rates of the distinct phases.

In the present test, the cell rates of A549 lung cancer cells indistinct phase of a cell cycle were observed. The results are shown inFIG. 2. Compared with the control group of A549 lung cancer cellsuntreated with the extract of Example 5, the experimental groups thereoftreated with the extract of Example 5 for 24, 48, and 72 hours all haveconsiderably increased cell rates in the G0/G1 phase. This demonstratesthat the extract of Example 5 blocks the cell cycle of A549 lung cancercells in the G0/G1 phase.

(2) Pi and Ki67 Antibody Co-Staining

A549 lung cancer cells were treated with the half maximal inhibitorydose (35 μl) of the extract of Example 5 for 72 hours, and thenunderwent PI and Ki67 antibody co-staining followed with flow cytometryto determine the cell rate of the A549 lung cancer cells in the G0phase. After statistics, the result is shown in FIG. 3.

As shown in FIG. 3, compared with the control group of A549 lung cancercells untreated with the extract of Example 5, the experimental groupthereof treated with the extract of Example 5 has considerably increasedcell rates in the G0 phase. This demonstrates that relatively more A549lung cancer cells do not accord with a cell cycle and they stay in theG0 phase (not division).

According to the results of the abovementioned test examples, it can beunderstood that the herbal medicinal composition and extract thereofprovided in the present invention can inhibit growth of cancer cells andmake the cancer cells stay in the G0 phase so as to block the continuousdivision of the cancer cells.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thescope of the invention as hereinafter claimed.

What is claimed is:
 1. An herbal medicinal composition for inhibitingcancer cells, comprising Angelicae Sinensis Radix in an amount of 1.5-6parts by weight, Ligustici Rhizoma in an amount of 1.5-6 parts byweight, Paeoniae Radix Rubra in an amount of 1.5-6 parts by weight,Rehmanniae Radix in an amount of 1.5-6 parts by weight, GlycyrrhizaeRadix in an amount of 1.5-6 parts by weight, Scutellariae Radix in anamount of 1,5-6 parts by weight, Chinese Wolfberry Root-bark in anamount of 2.5-10 parts by weight, the Peony Root-bark in an amount of2.5-10 parts by weight, and Atractylodis Rhizoma in an amount of 2-8parts by weight.
 2. A method for making an herbal medicinal extract forinhibiting cancer cells, comprising: mixing Angelicae Sinensis Radix,Ligustici Rhizoma, Paeoniae Radix Rubra, Rehmanniae Radix, GlycyrrhizaeRadix, Scutellariae Radix, Chinese Wolfberry Root-bark, Peony Root-bark,and Atractylodis Rhizoma to form a mixture; extracting the mixture withwater under heating to form an extract; and filtrating the extract toform the herbal medicinal extract, wherein the Angelicae Sinensis Radixin an amount of 1.5-6 parts by weight, the Ligustici Rhizoma in anamount of 1.5-6 parts by weight, the Paeoniae Radix Rubra in an amountof 1.5-6 parts by weight, the Rehmanniae Radix in an amount of 1.5-6parts by weight, the Glycyrrhizae Radix in an amount of 1.5-6 parts byweight, the Scutellariae Radix in an amount of 1.5-6 parts by weight,the Chinese Wolfberry Root-bark in an amount of 2.5-10 parts by weight,the Peony Root-bark in an amount of 2.5-10 parts by weight, and theAtractylodis Rhizoma in an amount of 2-8 parts by weight to remove dregsof the extract.